Stéphane Chevaliez

Long-term hepatitis B surface antigen (HBsAg) kinetics during nucleoside/nucleotide analogue therapy: Finite treatment duration unlikely

BACKGROUND & AIMS Information regarding long-term HBsAg kinetics during treatment with nucleoside/nucleotide analogues is limited. The aim of the present study was to assess whether finite nucleoside/nucleotide analogue treatment duration could be envisaged during the patients lifetime. METHODS Patients with chronic hepatitis B receiving different schedules of nucleoside/nucleotide analogues were followed for a median duration of 102 months, i.e., 8.5 years (interquartile range: 88-119 months). Long-term HBV DNA and HBsAg level kinetics were modeled in order to estimate time to clear HBsAg during therapy in patients with undetectable HBV DNA. RESULTS Antiviral therapy was associated with a slow but consistent reduction in the level of HBsAg in most of the patients. Three patterns of HBsAg level declines were identified: decline during both the detectable and undetectable HBV DNA phases; decline during the HBV DNA detectable period only; decline during the HBV DNA undetectable period only. The mean HBsAg titer at the time when HBV DNA became undetectable was 3.29 ± 0.49 Log₁₀ international units (IU)/ml, and the mean slope was -0.007 ± 0.007 Log₁₀ IU/month, i.e., an average decline of 0.084 Log₁₀ IU/year. The corresponding calculated median number of years needed to clear HBsAg was 52.2 years (interquartile range: 30.8-142.7). CONCLUSIONS This study, based on the very long-term follow-up of patients with chronic hepatitis B treated with potent nucleoside/nucleotide analogues, shows that HBsAg clearance is unlikely to occur during the patients lifetime, even if HBV replication is well controlled. Thus, lifetime therapy is required in the vast majority of HBV-infected patients.

Improvement of Hepatitis C Virus (HCV) Genotype Determination with the New Version of the INNO-LiPA HCV Assay

ABSTRACT Hepatitis C virus (HCV) isolates have been classified into six main genotypes. Genotyping methods, and especially the widely used line probe assay (LiPA), are frequently based on the 5′-untranslated region (5′UTR). However, this region is not appropriate for discriminating HCV strains at the subtype level and for distinguishing many genotype 6 samples from genotype 1. We investigated the capacity of a novel LiPA (Versant HCV Genotype 2.0 assay) based on the simultaneous detection of 5′UTR and Core regions for genotypes 1 and 6 to provide correct HCV genotypes (characterized with a phylogenetic analysis) in a set of HCV strains mainly encountered in Western countries. The improvement was assessed by comparing the results to those obtained with the previous version of the assay. Of the 135 tested samples, 64.7% were concordant for genotype group and subtype with sequencing reference results using the Versant HCV Genotype 2.0 assay versus 37.5% with the previous version. The yield was mainly related to a better characterization of genotype 1, since the accuracy, tested in 62 genotype 1 samples, increased from 45.2% with the first version to 96.8% with the new one. However, this new version necessitates a specific PCR and could no longer be used after 5′UTR PCR used for current HCV infection diagnosis. Moreover, the information provided by 5′UTR hybridization is not reliable for correctly identifying the diversity within genotypes 2 and 4. Thus, the Versant HCV Genotype 2.0 assay remains a useful tool for clinical practice when only the discrimination between major HCV genotypes is necessary.

New Virologic Tools for Management of Chronic Hepatitis B and C

Molecular biology techniques are routinely used to diagnose and monitor treatment of patients with chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. These tools can detect and quantify viral genomes and analyze their sequence to determine their genotype or subtype and to identify nucleotide or amino acid substitutions associated with resistance to antiviral drugs. They include real-time target amplification methods, which have been standardized and are widely used in clinical practice to diagnose and monitor HBV and HCV infections, and next-generation sequencing techniques, which are still restricted to research laboratories. In addition, new enzyme immunoassays can quantify hepatitis B surface and hepatitis C core antigens, and point-of-care tests and alternatives to biologic tests that require whole-blood samples obtained by venipuncture have been developed. We review these new virologic methods and their clinical and research applications to HBV and HCV infections.

Hepatitis C virus (HCV) genotype 1 subtype identification in new HCV drug development and future clinical practice.

Background With the development of new specific inhibitors of hepatitis C virus (HCV) enzymes and functions that may yield different antiviral responses and resistance profiles according to the HCV subtype, correct HCV genotype 1 subtype identification is mandatory in clinical trials for stratification and interpretation purposes and will likely become necessary in future clinical practice. The goal of this study was to identify the appropriate molecular tool(s) for accurate HCV genotype 1 subtype determination. Methodology/Principal Findings A large cohort of 500 treatment-naïve patients eligible for HCV drug trials and infected with either subtype 1a or 1b was studied. Methods based on the sole analysis of the 5′ non-coding region (5′NCR) by sequence analysis or reverse hybridization failed to correctly identify HCV subtype 1a in 22.8%–29.5% of cases, and HCV subtype 1b in 9.5%–8.7% of cases. Natural polymorphisms at positions 107, 204 and/or 243 were responsible for mis-subtyping with these methods. A real-time PCR method using genotype- and subtype-specific primers and probes located in both the 5′NCR and the NS5B-coding region failed to correctly identify HCV genotype 1 subtype in approximately 10% of cases. The second-generation line probe assay, a reverse hybridization assay that uses probes targeting both the 5′NCR and core-coding region, correctly identified HCV subtypes 1a and 1b in more than 99% of cases. Conclusions/Significance In the context of new HCV drug development, HCV genotyping methods based on the exclusive analysis of the 5′NCR should be avoided. The second-generation line probe assay is currently the best commercial assay for determination of HCV genotype 1 subtypes 1a and 1b in clinical trials and practice.

Analysis of Ribavirin Mutagenicity in Human Hepatitis C Virus Infection

ABSTRACT The addition of ribavirin to alpha interferon therapy significantly increases response rates for patients with chronic hepatitis C virus (HCV) infection, but ribavirins antiviral mechanisms are unknown. Ribavirin has been suggested to have mutagenic potential in vitro that would lead to “error catastrophe,” i.e., the generation of nonviable viral quasispecies due to the increment in the number of mutant genomes, which prevents the transmission of meaningful genetic information. We used extensive sequence-based analysis of two independent genomic regions in order to test in vivo the hypothesis that ribavirin administration accelerates the accumulation of mutations in the viral genome and that this acceleration occurs only when HCV replication is profoundly inhibited by coadministered alpha interferon. The rate of variation of the consensus sequence, the frequency of mutation, the error generation rate, and the between-sample genetic distance were measured for patients receiving ribavirin monotherapy, a combination of alpha interferon three times per week plus ribavirin, or a combination of alpha interferon daily plus ribavirin. Ribavirin monotherapy did not increase the rate of variation of the consensus sequence, the mutation frequency, the error generation rate, or the between-sample genetic distance. The accumulation of nucleotide substitutions did not accelerate, relative to the pretreatment period, during combination therapy with ribavirin and alpha interferon, even when viral replication was profoundly inhibited by alpha interferon. This study strongly undermines the hypothesis whereby ribavirin acts as an HCV mutagen in vivo.

Performance of the Abbott Real-Time PCR Assay Using m2000sp and m2000rt for Hepatitis C Virus RNA Quantification

ABSTRACT Quantification of hepatitis C virus (HCV) RNA is essential for the everyday management of chronic hepatitis C therapy. “Real-time” PCR techniques are potentially more sensitive than classical PCR techniques, are not prone to carryover contamination, and have a consistently wider dynamic range of quantification. Thus, they are rapidly replacing other technologies for routine quantification of HCV RNA. We extensively evaluated the intrinsic characteristics and clinical performance of the m2000sp-m2000rt Abbott real-time PCR platform for HCV RNA quantification. The study shows that the m2000sp-m2000rt platform is sensitive, specific, and precise; that the results are reproducible; and that the platform has a broad dynamic range of quantification. When comparing HCV RNA levels measured in the same individuals with the m2000sp-m2000rt platform and the third-generation branched-DNA assay, a trend toward a modest overestimation of HCV RNA levels was observed in the m2000sp-m2000rt platform in all genotypes except genotype 5. The differences, however, were unlikely to have any impact in clinical practice. In conclusion, our study shows that the Abbott m2000 real-time PCR system for HCV RNA quantification is sensitive, specific, and precise; that the results are reproducible; and that the platforms broad dynamic range of quantification is well suited to HCV RNA monitoring in the clinical setting.

Virological tools to diagnose and monitor hepatitis C virus infection

Approximately 200 million people are chronically infected with hepatitis C virus (HCV). Infection with HCV is curable by therapy, with the current standard treatment based on the combination of pegylated interferon-α and ribavirin. Viral eradication is achieved in approximately half of treated patients. In 2011 a new antiviral treatment based on a triple combination with a protease inhibitor will become available. Virological tools are essential to diagnose HCV infection but they have found their principal application in guiding treatment decisions and assessing the virological responses to therapy. These include the anti-HCV antibody assay, measurements of HCV core antigen and HCV viral load and HCV genotyping. The HCV RNA can be ideally assayed by a real-time assay with a limit of detection of 10-15 IU/mL. Monitoring of viral kinetics during the early phases of antiviral treatment is crucial in making treatment decisions such as early stopping rules and also in optimizing the treatment duration. The HCV genotype should be assessed before the start of treatment because it determines the treatment length and ribavirin dose and also offers prognostic information on treatment outcomes as certain genotypes respond more favourably to treatment.

The Cobas AmpliPrep‐Cobas TaqMan real‐time polymerase chain reaction assay fails to detect hepatitis C virus RNA in highly viremic genotype 4 clinical samples

five had submassive necrosis on liver biopsy. Five of these six patients underwent transplantation and all but one survived. One could not undergo transplantation due to development of fatal cholangitis. In conclusion, about 20% of patients with AIH have LF at initial presentation. A lower MELD score at admission ( 28), more severe hepatic fibrosis, and an early (within 4 days of steroid therapy) improvement or stabilization in serum bilirubin and INR identify those who are likely to respond to steroid therapy, and thus survive without LT. Our observations need further corroboration, but if confirmed, such a stratification strategy could optimize care of patients with AIH who present with LF.

Diagnosis and management of chronic viral hepatitis: Antigens, antibodies and viral genomes

Virological tools, including serological and molecular tools, are needed to diagnose chronic hepatitis B and C infections. They may also be useful to establish their prognosis, but they have found their principal application in guiding treatment decisions and assessing the virological responses to therapy. The goal of chronic hepatitis B therapy is to prevent progression of liver disease. This is achieved if HBV replication is durably abolished or significantly reduced. In HBeAg-positive patients, HBeAg clearance followed by the HBe seroconversion phase can be achieved. In HBeAg-negative patients, long-term antiviral suppression of viral replication is needed. The loss of HBsAg, eventually associated with an HBs seroconversion, is the most desirable endpoint of therapy but is rarely achieved. The efficacy endpoint of chronic hepatitis C treatment is the sustained virological response, defined by an undetectable HCV RNA in serum with a sensitive assay 24 weeks after the end of treatment. The HCV genotype and on-treatment viral kinetics can be used to tailor treatment dosages and duration.

Characterization of the dynamics of hepatitis B virus resistance to adefovir by ultra‐deep pyrosequencing

Hepatitis B virus (HBV) resistance to nucleoside/nucleotide analogs is frequent. Ultra‐deep pyrosequencing (UDPS) is a powerful new tool that can detect minor viral variants and characterize complex quasispecies mixtures. We used UDPS to analyze the dynamics of adefovir‐resistant HBV variants in patients with chronic HBV infection in whom adefovir resistance occurred during treatment. Amino acid substitutions known to confer resistance to adefovir were detected at baseline in most patients. The dynamics of adefovir‐resistant variants were complex and differed among patients as a result of evolving differences in variant fitness. UDPS analysis revealed successive waves of selection of HBV populations with single and multiple amino acid substitutions. Adefovir‐resistant variants were partially inhibited by lamivudine, but remained fit in its presence. Conclusion: Substitutions conferring HBV resistance to nucleoside/nucleotide analogs exist before treatment, and that the dynamics of adefovir‐resistant populations are much more complex and heterogeneous than previously thought and involve thus far unknown amino acid substitutions. The UDPS‐based approach described here is likely to have important implications for the assessment of antiviral drug resistance in research and clinical practice. (Hepatology 2013;53:890–901)